Directed cell migration in a gradient of chemoattractant is an important means by which mammalian cells can find sites of injury or infection in order to induce wound healing or immunological responses. The goal of the proposed research is to develop and test a model of the signal transduction mechanisms of chemotaxis at the molecular and cellular level. Specifically, we will use different live cell fluorescent imaging approaches to investigate the local signaling mechanisms at the leading edge that have been shown to include several second messengers and small G-proteins. The proposed studies will focus on the tyrosine kinase mediated chemotactic responses of CSF-1 stimulated macrophage and PDGF stimulated fibroblast cell lines. The specific aims of this project are supported by our construction of several fluorescent biosensors that can be used to track signaling processes in living migrating cells, by the building of a chemotaxis chamber that allows us to better manipulate extracellular gradients, by the development of a new type of evanescent wave microscope for chemotaxis studies as well as by the assembly of a near complete set of over a 100 known human small GTPases, together with their dominant negative and constitutively active mutants, for perturbation studies. Specifically, we will 1) determine the dynamic signaling role of PI(345)P3 and other second messengers during CSF1-R and PDGF-R initiated chemotaxis and relate signaling events to the changes in cell morphology and migration decisions, 2) determine the role of small GTPases by screening them for their effect on larnellipod extension, lipid signaling and chemotaxis and investigate their dynamic localization and activation during chemotactic migration, and 3) test a positive feedback hypothesis for chemotaxis based on localized PIP3 production by linking PI3-kinase, GEF and small GTPases into a positive feedback loop at the leading edge of chemotactic cells. The results from these studies will be used to generate a detailed model of the signaling machinery of chemotaxis. An understanding of this signaling process may lead to the identification of potential drug targets in the area of wound healing and immune responses.